Design, synthesis and immunological evaluation of CRM ₁₉₇ -based immunogens functionalized with synthetic scaffolds displaying a tumor-associated MUC1 glycopeptide

The development of effective vaccines against tumor-associated MUC1 (taMUC1) glycopeptide antigens remains a significant challenge due to their poor intrinsic immunogenicity. A key limitation in conjugate vaccine design lies in the structural alterations that occur upon carrier protein functionalization, which can reduce the accessibility of surface-conjugated antigens, ultimately compromising antigen presentation. In this study, we present a semi-synthetic vaccine platform in which taMUC1 glycopeptides are displayed on synthetic cyclopeptide scaffolds-configured either as monovalent or clustered tetravalent platforms-and subsequently grafted onto solvent-exposed amine residues of the CRM₁₉₇ protein <i>via</i> squaramide linkages. These conjugates were purified under denaturing conditions <i>via</i> reverse phase HPLC and evaluated <i>in vivo</i> through mouse immunization studies. Despite differences in antigen valency and glycopeptide loading per protein, both conjugates induced comparable levels of antigen-specific IgGs and CD4⁺/CD8⁺ T-cell activation when co-administered with the QS-21 adjuvant. Notably, although antibody titers were similar, post-immunization sera from mice immunized with the tetravalent conjugate plus the QS-21 adjuvant showed enhanced reactivity toward native taMUC1 expressed on MCF7 cancer cells, suggesting improved epitope recognition. These results highlight the impact of scaffold design, antigen display and adjuvantation on vaccine efficacy and establish a promising platform for the development of conjugate vaccines targeting weak tumor-associated antigens.